CN1218398A - Proopofol microdroplet formulations - Google Patents

Abstract

Formulations of phospholipid-coated microdroplets of propofol devoid of fats and triglycerides provide chronic sedation over extended periods of time without fat overload. Being free of nutrients that support bacterial growth, these microdroplet formulations are bacteriosatic and bactericidal (e.g. self-sterilizing) and thus have extended shelf life.

Description

Propofol microdroplet formulation

The invention relates to an intravenous anesthetic propofol pharmaceutical preparation.

Background of the invention

The present invention provides a formulation of the intravenous anesthetic drug Propofol (2,6-diisopropylphenol) in the form of phospholipid-coated microdroplets substantially completely free of fat or triglycerides. This formulation offers advantages for the long-term use of sedatives at a time when fat (triglyceride) overload is an important clinical factor. The formulations of the invention have also been shown to be bacteriostatic and bactericidal.

Propofol is a hydrophobic oil, ie an oil that is insoluble in water. It has been incorporated into vegetable oil emulsions to overcome its low water solubility and enable its use as an intravenous anesthetic. The clinically available product (PDR, 1995) is a sterile, pyrogen-free emulsion that is a white, 10% (W/V) emulsion of soybean oil in water. Sterile pharmaceutical compositions of Propofol and their use in the induction of anesthesia are described by Glen and James in U.S. Patents 4,056,635; 4,452,817 and 4,798,846. Propofol/soybean oil emulsion has been widely used for induction and/or maintenance of anesthesia, maintenance monitoring of anesthesia care, and for sedation in the intensive care unit (ICU). It produces a fast-acting anesthesia with short-term recovery.

Two concerns have been associated with the use of vegetable oils in 1% propofol/10% soybean oil emulsions: (1) hyperlipidemia in patients undergoing long-term ICU sedation, and (2) due to the high lipid content and lack of antimicrobial Preservatives create a risk of bacterial contamination.

The present invention provides phospholipid-coated propofol microdroplet formulations (MD-Propofol), which can be produced at a higher rate than current clinically effective products without soybean oil or other fats or triglycerides. Net load (weight/volume) of propofol delivered.

The formulation of propofol administered intravenously without the use of soybean oil, fat or triglycerides is an important feature of the present invention. Studies by Gottardis et al. 1989, De Sommer et al. 1990, Lindholm 1992, and Eddleston and Shelly 1991 have demonstrated that when 1% propofol/10% soybean oil emulsion is used as the sole agent for long-term ICU sedation Triglyceride overload becomes an important problem when sedatives are used. Administration of the propofol/soybean oil emulsion and the Intralipid(R) product on which it was based increased serum lipids in exactly the same way. It has been reported that in ICU sedation, if propofol/soybean oil emulsion is given to patients together with IV high nutrients, the fat overload will exceed the patient's ability to clear IV fat, resulting in "fat overload syndrome". The associated hyperlipidemia can lead to elevated bilirubin levels, "fatty liver", liver damage, and other adverse outcomes. It has also been noted that lipid tolerance can be reduced in critically ill patients caused by alterations in metabolic enzyme systems. Trials of 2% propofol emulsions delivering less fat per unit of propofol have been reported (Ewart et al., 1992; Dewabdre et al., 1994).

The absence of risk of bacterial growth in intravenously administered propofol formulations is a second important feature of the present invention. Commercially available products can grow bacteria and present a risk of bacterial contamination due to their high triglyceride content and lack of antimicrobial preservatives (Arduino et al., 1991; Sosis and Braverman, 1993; PDR, 1995). The phospholipid-coated Propofol microdroplet of the present invention does not contribute to bacterial growth, in fact, it is a bactericide.

Phospholipid-coated microdroplets of propofol coated with a stable monolayer of phospholipids are oily droplets of approximately 0.1 μm in diameter drug, my earlier U.S. Patents U.S. 4,622,219 and 4,725,442, the disclosures of which are hereby incorporated by reference It is described. Microdroplet formulations have been prepared from a number of compounds including methoxyflurane, isoflurane, and vitamin E, and the present invention provides microdroplet formulations of Propofol that allow fat-free administration of Propofol.

Brief description of attached drawings

The accompanying drawings are used to illustrate the present invention.

Figure 1 is a graphical representation of lecithin-coated propofol microdroplets.

Figure 2 is a graphical illustration of the duration of inhibition of the startle response as a function of dose of Propofol in microdroplets of the present invention compared to conventional Propofol/soybean oil emulsions.

Figure 3 is a graphical illustration of the duration of inhibition of the reinstatement response as a function of dose of propofol in microdroplet propofol of the present invention compared to conventional propofol/soybean oil emulsion.

Figure 4 is a graphical illustration of recovery time as a function of propofol dose for microdroplet propofol of the present invention compared to conventional propofol/soybean oil emulsion, and

Fig. 5 is after the propofol droplet of lecithin coating of the present invention is diluted 200 times (wherein curve A is the dilution of glucose/phosphate buffer saline, and curve B is the dilution of 5% bovine serum albumin) , a graphic illustration of its shrinkage kinetics determined by light scattering reduction.

Description of preferred implementation

The coating material of Propofol microdroplets can be selected from the lipids described in column 5-7 of my U.S. Patent No. 4,725,442 (introduced by reference) , especially the phospholipids described in Classes A, B and C . In addition, droplets can be coated with certain monoglycerides capable of forming oriented monolayers and bilayers in the presence of decane (Benz et al. Biochem. Biophys. Acta 394:323-334, 1975). Examples of useful monoglycerides include, but are not limited to, the following:

1-Monopalmitoyl-(rac)-glycerol (Monopalmitin)

1-Monocaprylyl (rac)-glycerol (glyceryl monocaprylate)

1-Monoleoyl (rac)-glycerol (18:1, cis-9) (monoolein)

1-Monostearyl (rac)-glycerol (monostearin)

Phosphatidylcholine (lecithin) is the most practical example. Egg phospholipid P123 from Pfanstiehl Laboratories Waukegan, IL is a pharmaceutical grade lecithin containing some phosphatidylethanolamine and cholesterol. In addition, stearoyl-, dimyristoyl- and dipalmitoyl-lecithins from Avanti Polar Lipids, Alabaseter, Alabama are of pharmaceutical grade, and they can be tested to show that the resulting product has the desired Use after physical stability.

The preparation of Propofol microdroplets requires strong mechanical agitation or high-speed shearing. A preferred method for preparing microdroplets of Propofol of the present invention on a laboratory scale is sonication with a probe sonicator. In industrial scale production, Microfluidization(R) (Microfluidics Corp., Newton, MA02164) is preferred. This method generates high shear forces through the collision of opposing jets of liquid. The apparatus is described by Mayhew et al., Acta Biochem. Biophys. 775:169-174, 1984. Another commercial scale processor includes, but is not limited to, a Gaulin and Rannie homogenizer (APV Gaulin/Rannie homogenizer, St, Paul, Minnesota).

The following examples further describe the invention. In these examples, an aqueous glucose/phosphate buffer consisting of 300 mM glucose, _{2 mM Na2HPO4} adjusted to pH 7.0 with HCl was used as the aqueous excipient for the microdroplet propofol formulation for preparation of Dilute and perform in vitro assays.

Measure the Propofol concentration in the product and the in vitro test by carrying out HPLC analysis on the methanol extract with a Beckman334 gradient liquid chromatography system, the parameters are as follows: mobile phase-methanol/water 65%/35% (v/v); Flow rate - 1.5 mL/min; UV detector (271 nm); Whatman Partisil 50DS-3 column (25 cm); injection volume - 50 μL.

Unless otherwise indicated, all parts and percentages stated herein are by weight per unit volume (w/v), where the volume as denominator represents the total volume of the system. The size of the diameter is expressed in millimeters (nm=10 ^-3 meters), microns (μm=10 ^-6 meters), nanometers (nm=10 ^-9 meters), or Angstrom units (=0.1nm). The volume is expressed in liter (L), milliliter (mL=10 ^-3 L) and microliter (μL=10 ^-6 L). Diluents are by volume. All temperatures are expressed in degrees Celsius. Compositions of the invention may comprise consisting essentially of or consisting of said matter, and methods may comprise consisting essentially of or consisting of said step having said matter.

Example 1

  (Propofol micro-droplet preparation)

At room temperature, lecithin (0.328 gm, egg phospholipid P123 Waukegan, IL from the Pfanstiehl laboratory), glucose/phosphate buffer (9.0 mL) and 2,6-diisopropylphenol (1.0 mL, Propofol, 97%, Aldrich Chemical Co.St.Louis, MO) placed in the glass test tube suspended on the water bath, through the thermal system-ultrasonic (Plainview, NY) Sonifier cell separator with the micro head of model W185D It's sonicated. Wear gloves and perform sonication in a fume hood during handling because propofol in its pure oil state is irritating. Sonication was performed at 60 watts for a total sonication time of 10 minutes (2 minutes on, 2 minutes off cycle to minimize heating of the sample). After sonication, the pH was adjusted to 7.0 with NaOH. This step produces lecithin-coated propofol droplets, which are a beige homogeneous suspension.

HPLC analysis determined the concentration of Propofol in the sample to be 68 mg/ml (6.8% w/v).

Particle size analysis was performed with a counter model N4MD submicron particle analyzer (Coulter Electronics, Hialeah, FL). Samples were diluted into propofol-saturated dextrose/phosphate buffer to minimize net release of propofol from the droplets. Analysis revealed a unimodal size distribution with a mean diameter of 164±54 (SD) nm.

Samples can also be determined by light microscopy in transmission mode with a Zeiss fluorescence microscope, the observed sample is a tightly packed suspension of 0.1-0.2 μm particles. Propofol droplets were observed as individual 0.1-0.2 μm particles undergoing Brownian motion following dilution with propofol saturating solution.

The formulation is stored at room temperature. During the 18-month period following the test, the formulation did not experience any settling or "emulsification", but a change in color or consistency. Importantly, there were no signs of bacterial or fungal growth.

Example 2

(Efficacy of general anesthesia in rats)

The effectiveness of the lecithin-coated propofol preparation (MD-propofol) in Example 1 and the commercial Diprivan® product in inducing anesthesia in laboratory rats was compared. Purchase Diprivan® (Diprivan® 1%, Propofol Injection 10 mg/ml, Emulsion for I.V. Administration, Stuart Pharmaceuticals), described by the manufacturer as a sterile, pyrogen-free emulsion containing 10 mg/ml in an aqueous vehicle ml propofol, 100mg/ml soybean oil, 12mg/ml lecithin. Store at room temperature as described by the manufacturer. Handle samples using aseptic technique.

Lecithin-coated propofol microdroplets containing 6.8% propofol and Diprivan® were injected into 150 g female CD laboratory rats immobilized in Decapicone® (Braintree Scientific, Braintree, MA) (Charles River Experimental Chamber, Wilmington, MA) in the tail vein. Injections of 6.8% (w/v) microdroplets of Propofol were performed in volumes of 10, 20, 30 or 50 [mu]L, and injections were completed within 2-3 seconds. The volume of 1% Diprivan(R) injected was 100, 200, 300 or 500 [mu]L, and the injection was completed within 5-15 seconds. The animals were observed during the injection and the time to loss of consciousness (time of anesthesia) was recorded. The rats were then removed from the Decapicone(R) and placed next to them and slapped loudly to test their startle response. A flinching response indicates light anesthesia; no overt response indicates deep anesthesia. The time to recover the startle response (startle reaction time) was recorded. The time to return to a stable response indicated by a spontaneous attempt to stand was also measured. The time elapsed from the onset of drug effect to the final return of the rats to baseline physical activity is referred to as the "full recovery time".

Tables 1 and 2 represent the dose-response data of the lecithin-coated microdroplets of Propofol and Diprivan(R), respectively, on laboratory rats. The table presents the mean values of: (a) the time required for the animal to respond unconsciously; (b) the time elapsed before the animal recovered a startle response to a loud slap; (c) the time before the animal recovered a stable response, as a function of dose. Elapsed time; and (d) time to full recovery, the table also shows the mortality rate.

Figures 2-4 show that MD-Propofol and Diprivan(R) have equivalent dose-response relationships for the four parameters.

Figure 2 graphically compares dose-response data for sustained startle responses elicited by MD-propofol and propofol/soybean oil emulsions. The dose-response curves for the two agents were identical, within experimental variation. The startle response represents the deepest degree of anesthesia measurable in nonoperative studies. The Student's test showed that there was no significant difference in the startle response duration between 12.6-13.3 mg/kg doses of MD-Propofol and Diprivan® (p=0.85).

Figures 3 and 4 compare the recovery time to stable response and the time to complete recovery of MD-Propofol and Diprivan® respectively, the dose-response curves of the two agents overlap, and the Student's test shows that when the dose is 12.6-13.3mg/kg There was no significant difference in response (p=0.50 and 0.42, respectively).

Tables 1 and 2 list propofol doses of 20-21 mg/kg to produce effective mortality, and the limited number of observations did not provide a statistical basis for differentiating the mortality of mice between the two groups.

Since the concentration of microdrop propofol was 6.8 times higher than that of the conventional propofol/soybean oil emulsion and because it was injected over a shorter period of time, the dilution effect of each formulation was investigated. Table 1 shows that administration of a 4-fold larger volume of microdroplet propofol at a dose of 12.6 mg/kg had no significant effect on any of the four anesthesia assays. Likewise, a 20 mg/kg dose of propofol/soybean oil emulsion at a 4-fold dilution had no significant effect.

Table 1 Rat dose-response for microdrop-Propofol MD-Propofol dosage mg/kg Unconscious time (minutes) Startled reaction time (minutes) Stable reaction time (minutes) Full recovery time (minutes) Mortality rate/person 4.2 NA 0.00 0.00 3.0±2.0 1/4 8.4 <1.0 2.5±5.0 4.7±7.1 9.8±9.2 0/4 12.6 <1.0 6.6±4.5 9.9±5.9 16.1±11.0 0/4 12.6* <1.0 3.7±4.7 4.5±4.5 9.7±8.8 0/5 21.0 <1.0 ** ** ** 3/5

*Dilute 4 times with pH7.0, 300mM glucose phosphate buffer.

**Manual cardiothoracic compression rescued 2 rats.

NA=never reached

Table 2 Dose-response of Diprivan® in laboratory rats Diprivan® dose (mg/kg) Unconscious time (minutes) Startled reaction time (minutes) Stable reaction time (minutes) Full recovery time (minutes) Mortality rate/person 6.7 <1.0 2.5±4.3 2.5±4.3 6.0±7.9 0/3 13.3 <1.0 8.8±2.6 10.1±1.1 18.4±6.3 0/5 20.0 <1.0 12.3±5.3 15.0±5.6 27.3±6.3 1/4 20.0* <1.0 14.0±4.6 16.7±2.5 25.3±5.0 2/3 33.3 <1.0 ** ** ** 4/5

*Dilute 4 times with pH7.00, 300mM glucose phosphate buffer.

**One survivor was thought to be due to subcutaneous extravasation during IV administration.

Example 3

(Release propofol into human plasma)

This test showed that both MD-Propofol and Diprivan(R) were able to release their propofol into human plasma in 30 seconds or less.

In a 10 x 75 mm borosilicate glass test tube, 6.8% microdrop propofol or (1%) propofol/(10%) soybean oil emulsion (Diprivan®) was diluted 200 aliquots with vortex mixing. Pour into human plasma (Continental Blood Bank, Miami, FL), and let it react for about 30 seconds or 10 minutes without stirring. Then, 210-250 μL aliquots were transferred to tared polyethylene centrifuge tubes and centrifuged in a Coriolis Microfuge for approximately 3 minutes. Propofol droplets migrate to the air-water interface, and the density of Propofol is 0.955. Similarly, the propofol/soybean oil emulsion migrated to the air-water interface, and the density of soybean oil was 0.916-0.922.

The tube was frozen, weighed and cut into two parts, which were weighed. The propofol solubles were then extracted with acidified alcohol, the methanol used to precipitate plasma proteins, which were removed by further centrifugation. As a control for this product, human plasma was also spiked with known amounts of Propofol and tested. It was calibrated to 100% (103±35%) extraction.

Table 3 gives the percentage of propofol released into human plasma after 29-31 seconds and 10 minutes. MD-Propofol and Diprivan(R) achieved a maximum release corresponding to 93% and 97% respectively within 32-34 seconds, with no significant difference between the two formulations.

Table 3. Comparison of MD-Propofol and Diprivan® dissolution percentages in human plasma preparation time after dilution Propofol dissolution percentage (%) Propofol undissolved percentage (%) MD-Propofol 34±3 seconds 92.7±8.9 7.3±8.9 Diprivan® 32±5 seconds 97.4±5.7 2.6±5.7 MD-Propofol 10 minutes 93.6±7.8 6.4±7.8 Diprivan® 10 minutes 99.5±1.3 0.5±1.3

Dilute MD-Propofol 200 times to 0.340mg/ml;

Diprivan(R) was diluted 200 times to 0.050 mg/ml.

Example 4

(Release from MD-Propofol monitored by light scattering)

The rate of contraction of propofol droplets accompanying propofol release in rats was measured by light scattering, as propofol droplets lose their highly refractive propofol cores and transform into liposomes or membrane lobes, which The 90° light scattering rate is reduced. The kinetics of Propofol droplet shrinkage was monitored by light scattering with a Perkin-Elmer MPF-3L fluorescence spectrophotometer equipped with a magnetic stirrer. The reaction was carried out in a clean 4-sided acrylic cuvette containing a Teflon-coated magnetic stirrer and filled with 2.0 ml of 5% bovine serum albumin solution (Sigma) as the Receptor or glucose/phosphate buffer served as controls. Human plasma cannot be used as a receptor for Propofol because of its intrinsic light scattering approximately equal to that of Propofol microdroplets.

Graph chart A of Fig. 5 is when adding embodiment 1 Propofol microdroplet (6.8%w/v) to dilute 200 times in the glucose/phosphate buffered saline of stirring picture. The introduction of the microdroplets results in an immediate increase in light scattering. As the decrease in propofol was observed within minutes of droplet release, the time of earliest signal detected was back-exposed to time zero to obtain its maximum at the time of dilution.

In Figure 4, curve B, the experiment was repeated in 5% bovine serum albumin. The graph shows that the earliest light scatter signal detected is only a small fraction of the signal observed in glucose/phosphate buffer, and subsequent scans are flat. The difference in the refractive index of the medium cannot explain the loss of light scattering. Thus, the release of propofol to the BAS medium was achieved within the two second mixing time tested.

Repeating the above experiment at higher sensitivities and graph speeds, we can observe a reduction in the final 1% of light scatter and measure a half-life of less than 1 second. The experiment was repeated several times with the same result. The initial amplitude observed for the BSA assay was only 4% of that for the glucose buffer assay. It was conservatively estimated that at least 96% of the release of propofol into the BAS was complete within two seconds.

Light scattering experiments showed that the microdroplets were able to release at least 94% of their propofol into the stirred glucose buffer. Several repetitions gave a half-life of 91±25 (SD) seconds. In this test, continued stirring was necessary for maximal release of Propofol from the droplets.

The complete incorporation of propofol in the micro-droplet into BSA takes less than 2 seconds. Such a short time for rapid release into plasma proteins is consistent with the time elapsed for monomeric propofol in the experiment of Example 2 to enter the brain from <1 minute to unconsciousness.

It is impractical to study Diprivan(R) release of propofol by means of light scattering because the Diprivan(R) particles do not shrink significantly, since vegetable oil is an important component before and after maximum propofol release.

Example 5

  (bacteriostatic and bactericidal activity of MD-Propofol)

The bacteriostatic and bactericidal activity tests of the droplet formulation (6.8% w/vprpofol) in Example 1 were carried out according to the description in USP 23, Chapter <71>, pages 1681-1689, 1995. Serial dilutions of E. coli SRB strains were made from stock growth suspensions (LB Broth Base, Gibco BRL, Cat. #12780-052, Lot #10E0252B) in sterile water. A 0.1 ml volume of the dilution was added to a 5 ml volume of 9:1 sterile growth medium to yield a 0.67% (w/v) concentration of Propofol. A 0.1ml volume of each bacterial dilution was also spread on the culture agar to determine the number of bacteria added to each test medium. After incubation at 37°C for 7 days, the test medium samples were spread on the culture agar to detect viable bacteria. And calculate the number of bacteria spread.

The above tests of MD-Propofol diluted to 0.67% (w/v) gave the following results: MD-Propofol was a bactericide for bacterial concentrations of 200 or less colony-forming units per milliliter; At bacterial concentrations of 500 to 1,000 colony-forming units per milliliter, MD-Propofol is a bacteriostatic agent.

Thus, the fat- and triglyceride-free microdroplet propofol formulations of the present invention are self-stable and offer the opportunity for a considerable shelf-life and less demanding manufacturing and packaging conditions.

The present invention has been described with respect to what are presently considered to be the most practical and preferred embodiments, and it is clear that the invention is not limited to the disclosed embodiments, but on the contrary, the invention covers every aspect included within the spirit and scope of the appended claims. modification and equivalent arrangement.

Cited additional literature Arduino, M.J.(1991) Infect.Control Hosp.Epidemiology 12(9):535-539 Eddleston, J.M, Shelly, M.P.(1991) Intensive Care Med.17(7):424-426Ewart, M.C., et al.(192)Anesthesia 47(2):146-148De Sommer,M.R.et al.(1990)Acta Anaesthesia Belgica 41(Ⅰ):8-12Dewandre,J.et al.(1994)Anaesthesia 49(Ⅰ):8 -12 Gottardis, M. et al. (1989) British J. Anaesthesia 62: 393-396 Lindholm, M. (1992) Minerva Anesthesiology 58 (10): 875-879 PDR (1995) entry, Stuart Pharmaceuticals, Wilmington, DE, in Physician' s Desk Reference, Medical Economics, Montvale, NJ, pp.2436-2441 Sosis, M.B., Braverman, B. (1993) Anesthes.Analges.77(4):766-768

Claims (10)

CLAIMS 1. Microdroplets from 200 angstroms to 1 micron in diameter, which are fat- and triglyceride-free and consist essentially of Propofol spheres coated with a phospholipid stabilization layer. 2. Microdroplets of about 200 angstroms to 1 micron in diameter consisting essentially of Propofol spheres coated with a phospholipid stabilization layer and free of oil to sustain bacterial growth. 3. A sterile, pyrogen-free injectable pharmaceutical composition consisting essentially of the microdroplets of claim 1 together with pharmaceutically acceptable injectable excipients. 4. The injectable pharmaceutical composition of claim 3, wherein the injectable excipient is an isotonic solution. 5. Microdroplets, produced by sonication, homogenization, microfluidization, or other methods including high-speed shearing, having a diameter of about 200 angstroms to 1 micron, having a polymeric stable core coated with a phospholipid coating coated, fat-free and triglyceride-free, wherein the ratio of the volume of propofol to the weight of the phospholipid coating is at least 1.0 ml/g, and wherein said microdroplets contain at least 3% w/v of propofol . 6. The droplet of claim 5 having a diameter of up to 10,000 Angstroms. 7. The droplet of claim 5, containing at least 5% w/v propofol. 8. Sterile, injectable pharmaceutical compositions consisting essentially of: (1) Microdroplets, about 200 angstroms to 1 micron in diameter, produced by sonication, homogenization, microfluidization, or other methods involving high-speed shear, and made of a propofol core stable to polymerization and A coating composition coated with a phospholipid film, wherein the ratio of the volume of propofol to the weight of the phospholipid film coating is at least 1.0ml/g, the composition contains at least 3% w/v of propofol and no fats and triglycerides; and (2) Pharmaceutical injectable carrier. 9. A bacteriostatic pharmaceutical composition consisting essentially of the following substances: (1) Microdroplets, about 200 angstroms to 1 micron in diameter, produced by sonication, homogenization, microfluidization, or other methods involving high-speed shear, and made of a propofol core stable to polymerization and A coating composition coated with a phospholipid film, wherein the ratio of the volume of propofol to the weight of the phospholipid film coating is at least 1.0ml/g, the composition contains at least 3% w/v of propofol and no fats and triglycerides; and (2) Pharmaceutical injectable carrier. 10. A fungicidal pharmaceutical composition consisting essentially of the following substances: (1) Microdroplets, about 200 angstroms to 1 micron in diameter, produced by sonication, homogenization, microfluidization, or other methods involving high-speed shear, and made of a propofol core stable to polymerization and A coating composition coated with a phospholipid film, wherein the ratio of the volume of propofol to the weight of the phospholipid film coating is at least 1.0ml/g, the composition contains at least 3% w/v of propofol and no fats and triglycerides; and (2) Pharmaceutical injectable carrier.

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